Peroxidase is an enzyme that catalyzes the oxidation of various compounds such as phenols and amines by peroxides. In addition, particular compounds have been termed pseudoperoxidases because they behave in a manner similar to the peroxidase enzyme by liberating oxygen from hydroperoxides and transferring the oxygen to certain acceptor compounds. Accordingly, the pseudoperoxidases are enzyme like in that they catalyze, or otherwise participate in, reactions between peroxides and oxidizable compounds. The pseudoperoxidases, which include hemoglobin and its derivatives, are regarded as peroxidatively active substances. For example, in the assay of urine for glucose the enzyme glucose oxidase, in the presence of oxygen, first converts the glucose in the urine to gluconic acid and hydrogen peroxide after which the peroxide enzyme which is included in the assay system catalyzes the interaction between the hydrogen peroxide (hydroperoxide) and an oxidizable dye, such as O-tolidine or tetramethylbenzidine, to cause the dye which is colorless in its reduced state to become colored thus providing a detectable response. The degree and intensity of the colored response are directly proportional to the amount of hydrogen peroxide generated by the glucose conversion, provided there is sufficient peroxidase present to catalyze the oxidation of the dye.
Similarly, a peroxidatively active substance such as hemoglobin or a derivative thereof can catalyze the interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase and catalyzes the interaction between the hydroperoxide and the oxidizable dye. The resulting interaction provides a detectable response, such as color transition, wherein the intensity of the response is indicative of the concentration of the peroxidatively active substance.
Creatinine is the end metabolite when creatine becomes creatine phosphate and is used as an energy source for muscle contraction. The creatinine produced is filtered by the kidney glomeruli and then excreted into the urine without reabsorption. The determination of creatinine in body fluids is useful for diagnosing muscle diseases or various kidney diseases such as nephritis and renal insufficiency. The first practical test for the determination of creatinine, known as the Jaffe method, involves the formation of the red-yellowish brown colored creatinine picrate by the bonding of picric acid and creatinine in an alkaline solution. A more recent method for creatinine determination is reported by Benedict and Behre in J. Biol. Chem., 113:515 (1936) which involves the reaction of 3,5-dinitrobenzoic acid with creatinine in an alkaline medium. Each of these reactions require a high pH, i.e. on the order of 12-13, in order to deprotonate the creatinine so that the system can operate properly. Strongly basic substances such as alkali and alkaline earth metal hydroxides are typically used to maintain a suitably high pH in these reagent systems. Operating at such high pH values presents various difficulties, especially when an absorbant carrier such as filter paper or a porous film is used as carrier for the reagent system. This is the case because upon introduction of the alkali, the carrier tends to become brittle and it is difficult to obtain even distribution of the alkali throughout the carrier matrix. Furthermore, when the reagents are applied to the carrier in the form of a solution followed by evaporating the solvent to leave a dry residue, the dried alkali does not readily solubilize when contacted with a fluid such as a urine sample which is being examined for creatinine concentration.
In U.S. Pat. No. 5,374,561 there is described a method for the detection of creatinine in an aqueous medium which involves contacting the medium suspected of containing creatinine with cupric ions in the presence of a hydroperoxide and a redox indicator which provides a colored response in the presence of oxygen free radicals. Also included in the creatinine reagent formulation disclosed in this patent is citrate to prevent urine components other than creatinine from complexing with the cupric ions. This patent also presents a series of equations which are believed to represent the reaction which results in a detectable response for the determination of creatinine: ##STR1##
In the foregoing scheme, reaction 1 represents the formation of the CuII. Creatinine complex from its resting state. Reaction 2 represents the oxidation of the TMB dye by the transfer of 1 electron from the TMB to the CuII. Creatinine complex to produce the non-reactive CuI form. Reaction 3 is the regeneration step whereby the CuI complex loses an electron to the peroxide to regenerate the CuII. An improved buffer system for this assay is disclosed in U.S. Pat. No. 5,733,787.
In order to increase the sensitivity of urinary assays and minimize the problem of high urine flow rates which result in urine dilution, analyte/creatinine ratios are used in urine analyte (e.g. protein) assays to normalize the urine concentration. Many clinically significant analytes are present in urine and urinalysis for them can be rendered more accurate by use of the creatinine ratio method. Among these analytes (sometimes referred to as the target analyte) are deoxypyridinoline, human serum albumin, drugs of abuse such as amphetamines, barbiturates and cocaine, clinically important protein markers such as prostate specific antigen; kidney disease proteins such as alpha-1-microglobulin, lactate dehydrogenase and N-acetyl-B-D-glucosamindase; pregnancy or fertility associated hormones such as human chorionic gonadotropin, follicle stimulating hormone and lutenizing hormone, markers of urinary tract infection such as Tamm-Horsfall protein or lipopolysaccharide, beta-2-microglobulin, amylase and chlamydial LPS.
In U.S. Pat. No. 5,173,431 there is disclosed a procedure for detecting proteins in fluids such as body fluids which involves contacting the fluid with a composition containing copper in a form capable of forming a copper/protein complex, which serves as a pseudoperoxidase, a peroxide and a redox indicator, which provides a detectable response when oxidized, together with an ionizable phosphate compound which can be phytic acid. This method does not require the presence of a citric acid since citric acid limits the formation of the copper/protein complex.